Method for counting red blood corpuscles in blood



Oct. 13, 1959 b m sso 2,908,160

METHOD FOR COUNTING RED BLOOD CORPUSCLES IN BLOOD Filed March 27, 1956Fig. 1

United States Patent NIETHOD FOR COUNTING RED BLOOD CORPUSCLES IN BLOODKarl Erik Harry Danielsson, Orehro, Sweden Application March 27, 1956,Serial No. 574,306

Claims priority, application Sweden March 29, 1955 2 Claims. (Cl. 73-61)The method hitherto almost exclusively used for the determination of thenumber of red blood corpuscles per unit of volume consists in a verygreat dilution of the blood and counting of the number of bloodcorpuscles in a thin layer of the diluted blood enclosed in a chamberformed between two glasses, a so called Biirker chamber. Said methodinvolves many sources of errors, is therefore inaccurate and is alsocircumstantial and time-consuming.

The present invention has for its object' to create a method for thedetermination of the number of red blood corpuscles, which is quickerthan the above-mentioned method and which, above all, gives moreaccurate results and eliminates personal errors to a great extent. Themethod according to the invention consists essentially therein, that atube filled with blood of the investigated sample is centrifuged,whereby the. blood corpuscles sedimentate and a border line is formed inthe tube between a lower column enriched with blood corpuscles and anupper blood plasma column practically devoid of blood corpuscles, thatthe centrifugal treatment is cut off after an accurately set time periodequal to a predetermined time period chosen for the apparatus used andbeing of such a short duration that the downward displacement of theborder line is considerably less than the displacement that would bereached as a final result of a very long period of centrifugaltreatment, and that the downward displacement of the border lineobtained for the sample is compared with displacement values obtained bythe centrifuging, under the same conditions, of a series of differentsamples having a number of red blood corpuscles per unit of volume,accurately determined by known methods, said values preferably beingused for calibrating a scale, on which the number of red bloodcorpuscles per unit of volume of the investigated sample can be directlyread off. The method is based upon the experience (verified by a greatnumber of comparative determinations) that the border linedrop valuesobtained after a short centrifugal treatment are closely related to thenumber of red blood corpuscles, whereas the final value obtained after avery long centrifugal treatment, i.e. the hematocrit value, does notgive any true information about the number of red blood corpuscles butmerely about their total volume, wherein the average size of the bloodcorpuscles is a factor.

The present invention also relates to a centrifugal apparatusparticularly suited for carrying out the abovementioned method and beingof such a construction as to eliminate personal errors in the handlingthereof, which errors would endanger the accuracy of the newblood-counting method.

As the centrifugal treatment should be cut off after a relatively shorttime, when the border line in the test tube is still dropping rapidly,it is obvious that the accuracy of the new method is dependent thereuponthat the centrifugal conditions are reproduceable with a high degree ofprecision. To this end, according to the invention, the centrifugalapparatus is provided with means ice for automatically maintainingconstant the centrifugal work accomplished upon the blood samples duringeach period of operation of the centrifugal apparatus. work isdetermined by many factors, out of which some are invariable, such asthe dimensions and the location of the test tube in the centrifugalapparatus, some factors may vary within tolerable limits, such as thefrequency and the voltage of the electric network, to which the drivemotor of the centrifugal apparatus is connected, the temperature etc.,whereas the time curve for the speed of rotation of the centrifugalapparatus during a period of operation is of a predominant importancefor the value of the drop of the border line in the test tube and forthe value of the number of blood corpuscles per unit of volume derivedtherefrom. Therefore, a main object of the invention is to provide thatthe time period of electric connection of the drive motor of thecentrifugal apparatus ,is held accurately constant and at apredetermined value.

According to the invention, in order accurately to provide the desiredtime period of operation the centrifugal apparatus is provided with atime switch for disconnecting the motor of the apparatus after apredetermined time of connection. Said time switch may be driven by thedrive motor of the apparatus, so that the switch is released when saidmotor has run a certain number of revolutions. Furthermore, it isimportant that the period of accelera tion and the period ofdeceleration are short in comparison to the period during which thecentrifugal apparatus rotates with full speed. In order to get a rapidacceleration a powerful motor should be chosen, and in order to get arapid deceleration, according to a feature of the invention, thecentrifugal apparatus is provided with a braking means, which is engagedautomatically and preferably simultaneously with the motor beingdisconnected.

The invention will be more closely described herein below, referencebeing had to the accompanying drawing, in which Fig. l is an elevationalView, partly in crosssection, and Fig. 2 is a top plan view, withcertain parts removed, of a preferred embodiment of the centrifugalapparatus forming part of the invention.

In the drawing, the numeral 11 designates a stationary cover, withinwhich an electric motor 13 with a vertical shaft is arranged. Attachedto the upper protruding end of the motor shaft is a horizontal plate 15,which in its turn carries an exactly centered, conical shell 17. Fourholders 19, in which test tubes 21 may be inserted, are evenly spacedaround and attached to the inside of said shell. In operation of theapparatus, test tubes are inserted in all of the holders, and in thosetest tubes that are not filled with blood, there is inserted a metal pinof wire with plug 39 for connection'to an electric network' of a fork 31which is fulcrumed about pivots 29 and pressed by a helical spring 33against the lower side of the sleeve 23. Said fork is connected by meansof a link 35 to a lever 37, the end of which extends out through thecover 11 so as to be operable manually. In

' the position shown, the lever 37 is depressed and the fork 31 is swungdown, compressing the spring 33, so that the brake consisting of themembers 23, 25, 27, is out of action and the top of the centrifuge isfree to rotate.

Mounted on the cover 11 are an electric connection Said of 220' volts, amain switch- 40, a control lamp 41-for indicatingconnectiontothenetwork, anadjustment knob 43 for adjusting thetapping-point on a resistance connected between the terminals of the,network, and a voltmeter 44 for indicatin'g'the voltagesupplied totheielectric metor 13. Connectedin series-withythe motoriis 'anotherswitch 45, which is located within the cover lil'. and is;

operable by means ofa push-rodI46-connected t o -thefOlk 31'.

pinion 47 which by means of a trainofgearsis coupledi' to a gear wheel49- carrying acan'1-disk5-l. The'g'ear ratio is 2000-: 1,-and thus thegear wheel 49 and the cam disk- 51 make one single complete revolutionwhen the l The blood used for the countingmethod according to theinvention'may be blood taken by a syringe from a cubital vein and maybediluted with one part of sodium citrate in four parts of blood. A testtube having a length-of. 110 mm. and an inner diameter-L2 mm. is filledwith said blood mixture to a predeterminedlevel, which may be marked byetched line or. may be the upper edge of the tube. The filled test tubeis inserted ina 'holder 19 in the centrifugal apparatus. Themainswitch40 is turned on, the lamp 41 being lighted'. By means of the knob 43 thevoltage is adjusted to a certain prescribed-value, e.g. 200 volts.

end position it is caught by the hitch 53, so-"that the operatorimmediately can leave hold of the lever; This causes-thedisengagement-of the brake and the simultaneous throwing-in of theswitch 45, whereby the motor is.

connected to the adjusted voltage of 200 volts. Themotor now acceleratesduring a period of say 20'seconds, aften which it reaches its fullspeed. When the-motor hasrun 2000 revolut-ions after its start, whichalways requires the same total time, say 45 seconds, the curve disk 51has turned one'com'plete-revolution and when the. steplthe rein. passes;the hitch53, thesame is let loose. ilnthisway the.

lever 37 is released and is raisedbythe spring 33, whereby. the motorcurrent is cut oil? and the brake is simultaneous: ly'engaged, so thatthe motor stops-in approximately 2 seconds; The test tube is nowremoved, and it proves. that the red blood corpuscles have sedimentated,so that there is a rather sharp border line at the middleofi the.

tube, which separates a lower column enriched with red blood corpusclesand an upper pale. column. of blood...

stance .as great volume. of blood plasmaasthe volume of bloodcorpuscles, and therefore the length of; said column is. practicallyindependent of the average size, of the blood corpuscles. .Therefore,had the centrifugal treatment, been continued, the border line wouldhave dropped iurtheron, and after a long time the lower column wouldhave contained practically nothing but blood corpuscles.

In this way the hematocrit value could have been obtained, which also isof interest in certain cases but which does not have any definiterelation to the number of red.

blood corpuscles. V

The length of the upper pale column in the test tubeis measured, and bycomparison with the result of earlier tests a figure on the number ofredbloodcells per cubic millimeter of the undiluted-blood is obtained..For instance, it it has been found-earlier, that a drop. of the;

border line of 31 mm. is obtained when blood havingQS millionsof redbloodcells'per cubic millimeter is treated.

in the apparatus above described and during the same time, then it isstated in a table or. in a diagram, that13;1

The lower end 'of the motor shaft carriesi a Then the operator depressesthe lever 37. Whenthe lever-reaches its lower mmfcorresponds to 5millions of blood corpuscles In the same way. corresponding values ofborder line drop and number of blood corpuscles are ascertained for aseries of diflerent blood samples, said number being determined asaccurately'as ever possible by using the best methods hitherto known,such as taking the average of a great number of determinations accordingto Biirker. These pairsof corresponding valuesare then tabulated orshown in'a diagram. Saidinterrelationshould be worked outbeforehandforthe apparatus used; it' is evident that it is valid onlyforcertainpredetermined conditions of centrifuging, such as time duration, speed.etc. Therefore these conditions must always betruly reproduced whenmaking. the-blood tests. 4 Q

The values obtainedduring the above-mentioned preliminary testscanpreferably be used for calibrating a scale for directly reading offthe number of-red blood cells per unit of volume of the bloodcontainedlin a test tube taken out of th'e centrifugal apparatus. whena; standard i'zed test tube is held against saids'cale with its bottom"resting against an end support associated with-the scale, the borderline in thetesttube points "to a figureon the scale stating thenumber of-millions of red blood-corpuscles in a; cubic rnil-limeter of theblood'beinginve'stigated. In accordance with the above-mentioned example,the=numeral--5 will be found on's'aid scale at a spot'situ- 'ated 31 mm.below the upper 'end'ofthescale, which end isflush-withthe upper edge ofthe test tube 'or the filling mark-engraved thereon. i i

T extensive statistical investigation has'proved that theabove-described -methodafifords "a' high degree of accuracy and that thewhite blood corpuscles (-leukocytes)- do not disturb the" result, noteven when their number is pathologically increased. The-method-israpidand canbe performed by untrained personnel, because it doesnot call torspecial care and attention. The course of centrifugal treatmentis'wholly automatic and the speed and' the time period are maintainedwith an accuracydriven by a-clockmovement or similar may be used. Itisrto be observed that thetime periods and the number of revolutionsmentioned above are merely given as examples, and that it is possible tostandardize the method and the apparatuson the basis of. other numericalvalues.

It is important, however, that the centrifugalapparatuses put upon themarket alford predetermined time periods and/or number of revolutionsthat are set for good, so

thatthe operatordoes not h'aveany possibility of readjusting the same,thereby, endangering the accuracy of thenew. blood-counting method. 7

. I claim: 0 I V, p I

. l. Amethod of determining the number ofred blood corpuscles per unitof volume of a blood sample, comprising: subjecting a tube filled withblood of thein vestigated sample to a centrifugal treatment, whereby'theI blood corpuscles sedimentate and a border line is formed in the tubebetween alower column enriched with red blood corpuscles and an uppercolumn of bloodpl-as'rna piractically'free'from red blood corpuscles,interrupting the centrifugaltreatment after an accurately determinedtime period equal to a time period prescribed for'thecentrifugalapparatus used, and of such-a short duration thattheresulting drop-ofsaidborder line is considerably less than the dropthat would be obtained as a final result after a prolonged, centrifugaltreatment, and comparing Instead of deriving the motor stopping andbraking impulse from the motor shaft, a time switch same conditions of aseries of diiferent blood samples having a number of red bloodcorpuscles per unit of volume, determined accurately by previously knownmethods.

2. A method as claimed in claim 1, in which comparison is made betweenthe border line drop obtained for the investigated sample and a scalecalibrated with guidance of the drop values obtained for the known bloodsamples, whereby the number of red blood corpuscles per unit ofReferences Cited in the file of this patent volume of the investigatedsample can be directly read 0E. 1 2,741,913

UNITED STATES PATENTS Allison i Nov. 22, Garver Feb. 19, Levy May 28,Tholl Apr. 1, Worley Dec. 6, Scheflier Dec. 27, Dovas Apr. 17

